Calcium carbonate (CaCO3) nanocrystals derived from cockle shells emerge to present a good concert in bone tissue engineering because of their potential to mimic the composition, structure, and properties of native bone. The aim of this study was to evaluate the biological response of CaCO3 nanocrystals on hFOB 1.19 and MC3T3 E-1 osteoblast cells in vitro. Cell viability and proliferation were assessed by MTT and BrdU assays, and LDH was measured to determine the effect of CaCO3 nanocrystals on cell membrane integrity. Cellular morphology was examined by SEM and fluorescence microscopy. The results showed that CaCO3 nanocrystals had no toxic effects to some extent. Cell proliferation, alkaline phosphatase activity, and protein synthesis were enhanced by the nanocrystals when compared to the control. Cellular interactions were improved, as indicated by SEM and fluorescent microscopy. The production of VEGF and TGF-1 was also affected by the CaCO3 nanocrystals. Therefore, bio-based CaCO3 nanocrystals were shown to stimulate osteoblast differentiation and improve the osteointegration process.
Suspension mononuclear cells (MNCs) can be differentiated into osteoblasts with the induction of ascorbic acid and β-glycerophosphate. The aim of this study was to determine the ability of suspension MNCs to differentiate into osteoblasts using ascorbic acid only.
A hydroxyapatite scaffold is a suitable biomaterial for bone tissue engineering due to its chemical component which mimics native bone. Electronic states which present on the surface of hydroxyapatite have the potential to be used to promote the adsorption or transduction of biomolecules such as protein or DNA. This study aimed to compare the morphology and bioactivity of sinter and nonsinter marine-based hydroxyapatite scaffolds. Field emission scanning electron microscopy (FESEM) and micro-computed tomography (microCT) were used to characterize the morphology of both scaffolds. Scaffolds were co-cultured with 5 × 104/cm2 of MC3T3-E1 preosteoblast cells for 7, 14, and 21 days. FESEM was used to observe the cell morphology, and MTT and alkaline phosphatase (ALP) assays were conducted to determine the cell viability and differentiation capacity of cells on both scaffolds. Real-time polymerase chain reaction (rtPCR) was used to identify the expression of osteoblast markers. The sinter scaffold had a porous microstructure with the presence of interconnected pores as compared with the nonsinter scaffold. This sinter scaffold also significantly supported viability and differentiation of the MC3T3-E1 preosteoblast cells (p < 0.05). The marked expression of Col1α1 and osteocalcin (OCN) osteoblast markers were also observed after 14 days of incubation (p < 0.05). The sinter scaffold supported attachment, viability, and differentiation of preosteoblast cells. Hence, sinter hydroxyapatite scaffold from nacreous layer is a promising biomaterial for bone tissue engineering.
Morphological surface modifications have been reported to enhance the performance of biomedical implants. However, current methods of introducing graded porosity involves postprocessing techniques that lead to formation of microcracks, delamination, loss of fatigue strength, and, overall, poor mechanical properties. To address these issues, we developed a microwave sintering procedure whereby pure titanium powder can be readily densified into implants with graded porosity in a single step. Using this approach, surface topography of implants can be closely controlled to have a distinctive combination of surface area, pore size, and surface roughness. In this study, the effect of various surface topographies on in vitro response of neonatal rat calvarial osteoblast in terms of attachment and proliferation is studied. Certain graded surfaces nearly double the chance of cell viability in early stages (∼one month) and are therefore expected to improve the rate of healing. On the other hand, while the osteoblast morphology significantly differs in each sample at different periods, there is no straightforward correlation between early proliferation and quantitative surface parameters such as average roughness or surface area. This indicates that the nature of cell-surface interactions likely depends on other factors, including spatial parameters.
Hydroxyapatite (Ca10(PO4)6(OH)2) is widely investigated as an implantable material for hard tissue restoration due to its osteoconductive properties. However, hydroxyapatite in bulk form is limited as its mechanical properties are insufficient for load-bearing orthopedic applications. Attempts have been made to improve the mechanical properties of hydroxyapatite, by incorporating ceramic fillers, but the resultant composite materials require high sintering temperatures to facilitate densification, leading to the decomposition of hydroxyapatite into tricalcium phosphate, tetra-calcium phosphate and CaO phases. One method of improving the properties of hydroxyapatite is to incorporate bioactive glass particles as a second phase. These typically have lower softening points which could possibly facilitate sintering at lower temperatures. In this work, a bioactive glass (SiO2-CaO-ZnO-Na2O-TiO2) is incorporated (10, 20 and 30 wt%) into hydroxyapatite as a reinforcing phase. X-ray diffraction confirmed that no additional phases (other than hydroxyapatite) were formed at a sintering temperature of 560 ℃ with up to 30 wt% glass addition. The addition of the glass phase increased the % crystallinity and the relative density of the composites. The biaxial flexural strength increased to 36 MPa with glass addition, and there was no significant change in hardness as a function of maturation. The pH of the incubation media increased to pH 10 or 11 through glass addition, and ion release profiles determined that Si, Na and P were released from the composites. Calcium phosphate precipitation was encouraged in simulated body fluid with the incorporation of the bioactive glass phase, and cell culture testing in MC-3T3 osteoblasts determined that the composite materials did not significantly reduce cell viability.
Far-flung evolution in tissue engineering enabled the development of bioactive and biodegradable materials to generate biocomposite nanofibrous scaffolds for bone repair and replacement therapies. Polymeric bioactive nanofibers are to biomimic the native extracellular matrix (ECM), delivering tremendous regenerative potentials for drug delivery and tissue engineering applications. It's been known from few decades that Zinc oxide (ZnO) nanoparticles are enhancing bone growth and providing proliferation of osteoblasts when incorporated with hydroxyapatite (HAp). We attempted to investigate the interaction between the human foetal osteoblasts (hFOB) with ZnO doped HAp incorporated biocomposite poly(L-lactic acid)-co-poly(ε-caprolactone) and silk fibroin (PLACL/SF) nanofibrous scaffolds for osteoblasts mineralization in bone tissue regeneration. The present study, we doped ZnO with HAp (ZnO(HAp) using the sol-gel ethanol condensation technique. The properties of PLACL/SF/ZnO(HAp) biocomposite nanofibrous scaffolds enhanced with doped and blended ZnO/HAp were characterized using Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Contact angle and Tensile studies to determine the morphology, functionality, wettability and stability. The in vitro study results showed that the addition of ZnO and HAp enhances the secretion of bone mineral matrix (98%) with smaller fiber diameter (139.4 ± 27 nm) due to the presence of silk fibroin showing potential tensile properties (322.4%), and increased the proliferation of osteoblasts for bone tissue regeneration.
The multipotency of stromal cells has been studied extensively. It has been reported that mesenchymal stromal cells (MSC) are capable of differentiating into cells of multilineage. Different methods and reagents have been used to induce the differentiation of MSC. We investigated the efficacy of different growth factors in inducing MSC differentiation into neurons.
Bone is a specialized connective tissue that functions as the load-bearing structure of the body. Free radicals may affect bone remodeling by regulating osteoclast activity in either the physiological or pathological condition. Vitamin E, a lipid-soluble antioxidant, has been demonstrated to offer protection against osteoporosis and to improve the bone material and structure of animal models. The aim of this study was to observe and compare the effects of alpha-tocopherol (alpha-tocopherol), delta-tocotrienol (delta-tocotrienol), and gamma-tocotrienol (gamma-tocotrienol) on the static and dynamic bone histomorphometric parameters in normal male rats. Thirty-two normal Sprague-Dawley male rats aged 3 months and weighing 200-250 g were randomly divided into four groups. The control group was supplemented with oral gavages of olive oil (vehicle), whereas the alpha-tocopherol, delta-tocotrienol, and gamma-tocotrienol groups were given oral gavages of 60 mg/kg alpha-tocopherol, delta-tocotrienol, and gamma-tocotrienol, respectively. The rats were injected twice with calcein to fluorochrome-label the bones. After 4 months of treatment, the rats were killed, and the left femurs were dissected out and prepared for bone histomorphometry. Both the static and dynamic parameters of the vitamin E-treated groups were better than those of the normal control group. Among the vitamin E-treated groups, the tocotrienol groups showed better histomorphometry results compared to the α-tocopherol group, with the γ-tocotrienol group demonstrating the best effects on both sets of parameters. We concluded that vitamin E can promote bone formation in normal rats, with gamma-tocotrienol being the most potent form of vitamin E.
Phytoestrogens have attracted considerable attention for their potential in the prevention of postmenopausal osteoporosis. Recently, a phytoestrogen-rich herbal plant, Marantodes pumilum var. alata (Blume) Kuntze was reported to protect against bone loss in ovariectomized rat. However, the bioactive compound responsible for these effects and the underlying mechanism were not known. Through bioassay-guided isolation, demethylbelamcandaquinone B (Dmcq B) was isolated and identified from Marantodes pumilum var. alata leaf extract. In terms of its bone anabolic effects, Dmcq B was at par with 17β-estradiol (E2), in promoting the proliferation, differentiation and mineralization of osteoblast cells. Dmcq-B increased early differentiation markers, collagen content and enzymatic ALP activity. It was demonstrated to regulate BMP2 signaling pathway which further activated the transcription factor, osterix. Subsequently, Dmcq B was able to increase the osteocalcin expression which promoted matrix mineralization as evidenced by the increase in calcium deposition. Dmcq B also reduced the protein level of receptor activator of NF-κβ ligand (RANKL) and promoted osteoprotegerin (OPG) protein expression by osteoblast cells, therefore hastening bone formation rate by decreasing RANKL/OPG ratio. Moreover, Dmcq B was able to increase ER expression, postulating its phytoestrogen property. As the conclusion, Dmcq B is the active compound isolated from Marantodes pumilum var. alata leaves, regulating osteoanabolic activities potentially through the BMP2 and ER signaling pathways.
Stem cells (SCs) are capable of self-renewal and multilineage differentiation. Human mesenchymal stem cells (MSCs) and haematopoietic stem cells (HSCs) which can be obtained from multiple sources, are suitable for application in regenerative medicine and transplant therapy. The aim of this review is to evaluate the potential of genomic and proteomic profiling analysis to identify the differentiation of MSCs and HSCs towards osteoblast and odontoblast lineages. In vitro differentiation towards both of these lineages can be induced using similar differentiation factors. Gene profiling cannot be utilised to confirm the lineages of these two types of differentiated cells. Differentiated cells of both lineages express most of the same markers. Most researchers have detected the expression of genes such as ALP, OCN, OPN, BMP2 and RUNX2 in osteoblasts and the expression of the DSPP gene in odontoblasts. Based on their cell-type specific protein profiles, various proteins are differentially expressed by osteoblasts and odontoblasts, except for vimentin and heterogeneous nuclear ribonucleoprotein C, which are expressed in both cell types, and LOXL2 protein, which is expressed only in odontoblasts.
In this study the surface properties of two particulate coral and polyhydroxybutrate (PHB) were studied in order to characterize them prior to use in composite production. Coral powder and PHB particle were evaluated using scanning electron microscopy and confocal laser scanning microscopy, to measure surface porosity and pores size. The results showed that coral powder has multiple pleomorphic micropores cross each others give appearance of micro-interconnectivity. Some pore reached to 18 microm with an average porosity of 70%. PHB revealed multiple different size pores extended to the depth, with an average some times reach 25 microm and porosity 45%. These findings demonstrate that both coral and PHB have excellent pores size and porosity that facilitate bone in growth, vascular invasion and bone development. We believe that incorporation of coral powder into PHB will make an excellent composite scaffold for tissue engineering.
This study was designed to evaluate the ability of natural coral implant to provide an environment for marrow cells to differentiate into osteoblasts and function suitable for mineralized tissue formation. DNA content, alkaline phosptatase (ALP) activity, calcium (Ca) content and mineralized nodules, were measured at day 3, day 7 and day 14, in rat bone marrow stromal cells cultured with coral discs glass discs, while cells alone and coral disc alone were cultured as control. DNA content, ALP activity, Ca content measurements showed no difference between coral, glass and cells groups at 3 day which were higher than control (coral disc alone), but there were higher measurement at day 7 and 14 in the cell cultured on coral than on glass discs, control cells and control coral discs. Mineralized nodules formation (both in area and number) was more predominant on the coral surface than in control groups. These results showed that natural coral implant provided excellent and favorable situation for marrow cell to differentiate to osteoblasts, lead to large amount of mineralized tissue formation on coral surface. This in vitro result could explain the rapid bone bonding of coral in vivo.
The utilisation of hydroxyapatite and collagen as bioactive coating materials could enhance cells attachment, proliferation and osseointegration. However, most methods to form crystal hydroxyapatite coating do not allow the incorporation of polymer/organic compound due to production phase of high sintering temperature. In this study, a polydopamine film was used as an intermediate layer to immobilise hydroxyapatite-collagen without the introduction of high sintering temperature. The surface roughness, coating adhesion, bioactivity and osteoblast attachment on the hydroxyapatite-collagen coating were assessed as these properties remains unknown on the polydopamine grafted film. The coating was developed by grafting stainless steel 316L disks with a polydopamine film. Collagen type I fibres were then immobilised on the grafted film, followed by the biomineralisation of hydroxyapatite. The surface roughness and coating adhesion analyses were later performed by using AFM instrument. An Alamar Blue assay was used to determine the cytotoxicity of the coating, while an alkaline phosphatase activity test was conducted to evaluate the osteogenic differentiation of human fetal osteoblasts on the coating. Finally, the morphology of cells attachment on the coating was visualised under FESEM. The highest RMS roughness and coating adhesion were observed on the hydroxyapatite-collagen coating (hydroxyapatite-coll-dopa). The hydroxyapatite-coll-dopa coating was non-toxic to the osteoblast cells with greater cells proliferation, greater level of alkaline phosphate production and more cells attachment. These results indicate that the immobilisation of hydroxyapatite and collagen using an intermediate polydopamine is identical to enhance coating adhesion, osteoblast cells attachment, proliferation and differentiation, and thus could be implemented as a coating material on orthopaedic and dental implants.
Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be selectively isolated and induced to express bone phenotype in vitro. The osteoblastic progenitor can be estimated by counting the number of cells attach using the haemacytometer. This study was undertaken to test the hypothesis that human aging is associated with a significant change on the number of osteoblastic progenitors in the bone marrow. Bone marrow aspirates were harvested from 38 patients, 14 men (age 11-70) and 24 women (age 10-70) and cultured in F12: DMEM (1:1). In total 15 bone marrow samples have been isolated from patients above 40 years old (men/women) of age. Fourteen (93.3%) of this samples failed to proliferate. Only one (6.7%) bone marrow sample from a male patient, aged 59 years old was successfully cultured. Seventy percent (16/23) of the samples from patient below than 40 years old were successfully cultured. However, our observation on the survival rate for cells of different gender from patient below 40 years old does not indicate any significant difference. From this study, we conclude that the growth of bone marrow stromal cells possibly for bone engineering is better from bone marrow aspirates of younger patient.
Bioceramic nanoparticles with high specific surface area often tend to agglomerate in the polymer matrix, which results in undesirable mechanical properties of the composites and poor cell spreading and attachment. In the present work, bredigite (BR) nanoparticles were modified with an organosilane coupling agent, 3-glycidoxypropyltrimethoxysilane (GPTMS), to enhance its dispersibility in the polymer matrix. The polyhydroxybutyrate-co-hydroxyvaletare (PHBV) nanofibrous scaffolds containing either bredigite or GPTMS-modified bredigite (G-BR) nanoparticles were fabricated using electrospinning technique and characterized using scanning electron microscopy, transmission electron microscopy, and tensile strength. Results demonstrated that modification of bredigite was effective in enhancing nanoparticle dispersion in the PHBV matrix. PHBV/G-BR scaffold showed improved mechanical properties compared to PHBV and PHBV/BR, especially at the higher concentration of nanoparticles. In vitro bioactivity assay performed in the simulated body fluid (SBF) indicated that composite PHBV scaffolds were able to induce the formation of apatite deposits after incubation in SBF. From the results of in vitro biological assay, it is concluded that the synergetic effect of BR and GPTMS provided an enhanced hFob cells attachment and proliferation. The developed PHBV/G-BR nanofibrous scaffolds may be considered for application in bone tissue engineering.
Interconnected porous tricalcium phosphate ceramics are considered to be potential bone substitutes. However, insufficient mechanical properties when using tricalcium phosphate powders remain a challenge. To mitigate these issues, we have developed a new approach to produce an interconnected alpha-tricalcium phosphate (α-TCP) scaffold and to perform surface modification on the scaffold with a composite layer, which consists of hybrid carbonate apatite / poly-epsilon-caprolactone (CO3Ap/PCL) with enhanced mechanical properties and biological performance. Different CO3Ap combinations were tested to evaluate the optimal mechanical strength and in vitro cell response of the scaffold. The α-TCP scaffold coated with CO3Ap/PCL maintained a fully interconnected structure with a porosity of 80% to 86% and achieved an improved compressive strength mimicking that of cancellous bone. The addition of CO3Ap coupled with the fully interconnected microstructure of the α-TCP scaffolds coated with CO3Ap/PCL increased cell attachment, accelerated proliferation and resulted in greater alkaline phosphatase (ALP) activity. Hence, our bone substitute exhibited promising potential for applications in cancellous bone-type replacement.
Calcium silicate (CaSiO3, CS) ceramics are promising bioactive materials for bone tissue engineering, particularly for bone repair. However, the low toughness of CS limits its application in load-bearing conditions. Recent findings indicating the promising biocompatibility of graphene imply that graphene can be used as an additive to improve the mechanical properties of composites. Here, we report a simple method for the synthesis of calcium silicate/reduced graphene oxide (CS/rGO) composites using a hydrothermal approach followed by hot isostatic pressing (HIP). Adding rGO to pure CS increased the hardness of the material by ∼40%, the elastic modulus by ∼52%, and the fracture toughness by ∼123%. Different toughening mechanisms were observed including crack bridging, crack branching, crack deflection, and rGO pull-out, thus increasing the resistance to crack propagation and leading to a considerable improvement in the fracture toughness of the composites. The formation of bone-like apatite on a range of CS/rGO composites with rGO weight percentages ranging from 0 to 1.5 has been investigated in simulated body fluid (SBF). The presence of a bone-like apatite layer on the composite surface after soaking in SBF was demonstrated by X-ray diffraction (XRD) and field emission scanning electron microscopy (FESEM). The biocompatibility of the CS/rGO composites was characterized using methyl thiazole tetrazolium (MTT) assays in vitro. The cell adhesion results showed that human osteoblast cells (hFOB) can adhere to and develop on the CS/rGO composites. In addition, the proliferation rate and alkaline phosphatase (ALP) activity of cells on the CS/rGO composites were improved compared with the pure CS ceramics. These results suggest that calcium silicate/reduced graphene oxide composites are promising materials for biomedical applications.